The metaphase to anaphase transition is a complex and critical step in mitosis. Multiple regulatory signals impinge on the decision to initiate sister chromatid separation, including those involved in nonessential checkpoint regulation as well as those that are essential components of cell cycle timing machinery. Defects in any of these mechanisms can lead to abnormal chromosome segregation resulting in aneuploidy or cell death. The identity of the components of these pathways and the mechanisms by which they act are essentially unknown. The long term goal of this research is to define, in molecular detail, the mechanisms that regulate the metaphase to anaphase transition in Aspergillus nidulans. This information will be of general interest because many of the fundamental events of mitosis are conserved in Aspergillus. This proposal focuses on the function of BimA, an evolutionarily conserved protein that is required for the onset of anaphase and is located at the spindle pole body (SPB). These facts, and preliminary genetic studies indicating that BimA interacts with mitotic regulatory kinases, make BimA a promising candidate for a component of the essential pathway regulating the onset of anaphase. The proposed research has three major aims: 1. Determine the role of BimA in the function of the p34cdc2 and NimA mitotic kinases. New and existing immunological reagents will be used to determine the activity, accumulation, and subcellular localization of p34cdc2/cyclin B and NimA in bimA- mutants. Specific mutations in p34cdc2/cyclin B and in nimA will be made and characterized to test the hypothesis that BimA promotes the onset of anaphase by regulating the function of p34cdc2. 2. Determine the role of BimA structure and accumulation in BimA localization and function. Site-specific and random mutations in bimA will be made in vitro and studied in vivo for their effects on BimA localization and function. Conserved and unique BimA sequences will be tested for their ability to confer BimA-specific localization to heterologous proteins. In vivo 32P labelling and BimA immunoprecipitation will be used to determine whether BimA is phosphorylated. 3. Determine the role of SPB and mitotic apparatus components in BimA localization. Mutations or drugs that perturb microtubule assembly will be used to determine the role of microtubules in BimA localization. Proteins that physically interact with BimA will be identified by coimmunoprecipitation or chemical crosslinking studies. High resolution in situ localization of BimA will be performed by immunoelectron microscopy using existing affinity purified antisera.